This project focuses on the physiologically critical functions of NF-kappaB proteins and their regulators. To investigate such functions we assess biologic responses in vivo with mouse models engineered to lack components of the NF-kappaB transcription factor family or of their regulators. Regulators of interest include the classical inhibitory IkappaB proteins, the non-inhibitory IkappaB family protein Bcl-3, as well as several proximal components of signaling pathways that lead to activation of NF-kappaB. Research is based on the discovery of specific biologic defects (especially of the immune system) in mice rendered deficient for various NF- kappaB proteins or their regulators. The ultimate goal is to identify critical molecular targets of the NF-kappaB factors and their regulators in specific immune responses/diseases and to identify the essential signaling pathways that activate the factors in these situations. We have shown previously that mice deficient in both NF-kappaB1 and NF-kappaB2 subunits are completely blocked in the development of mature osteoclasts and mature B cells. Using adoptive transfer techniques we further demonstrated that these developmental blocks are intrinsic to the respective cell lineages, i.e. osteoclasts and B cells. B cells are blocked at the transitional stage in spleen, and consequently no recirculating mature B cells are generated. The block is due in large part to loss by apoptosis of all mutant transitional B cells. We have now demonstrated that this loss can be overcome by introduction of Bcl-2 into the mutant B cells, either by introduction of a transgene into the mouse genome or by lentivirus-mediated transduction of bone marrow cells. The rescued B cells progressed further, but still failed to fully mature phenotypically and functionally, indicating roles for NF-kappaB1 and NF-kappaB2 not only in survival but also in other aspect of developmental progression. We have now confirmed this conclusion with other experimental approaches. Our previous research also revealed that BAFF and its primary receptor are responsible, at least in part, for activating NF-kappaB in normal transitional B cells. The BAFF receptor engages primarily the non-canonical pathway of activation for NF-kappaB, a pathway in which the NF-kappaB2 precursor protein p100 is processed to p52, leading to nuclear translocation of RelB/p52 dimers. We also demonstrated previously that NF-kappaB2 processing in response to lymphotoxin beta receptor stimulation in stromal cells was essential for the development of follicular dendritic cell networks, B cell follicles, germinal centers and lymph node formation. Of note, loss of NF-kappaB2 or loss of the NF-kappaB regulator Bcl-2 in mice resulted in similar phenotypes with respect to the structure secondary lymphoid organs, suggesting that these two proteins lie on a linear pathway in regulating some critical genes in stromal cells. However, these proteins appear also to have redundant activities, as mice deficient in both Bcl-3 and NF-kappaB2 have unexpected phenotypes in addition to those seen in single knockouts. In particular, these doubly-deficient mice develop severe multi-organ inflammation very early in life. Another project is related to the recent discovery that NF-kappaB activated via the classical pathway may have a necessary role to play in development of insulin resistance and thus diabetes type 2. We have now determined that NF-kappaB?s role in this disease may be more extensive than initially surmised, as mice lacking NF-kappaB2, a subunit not normally associated with the classical activation pathway or with immediate inflammatory responses, is also critical for development of obesity-induced insulin resistance.